Sparc and methods of use thereof

ABSTRACT

The invention provides methods for predicting or determining the response of a mammalian tumor to a chemotherapeutic agent and for treating a mammalian tumor comprising detecting and quantifying the SPARC protein or RNA in a sample isolated from the mammal. The invention further provides kit for predicting the response of a mammalian tumor to a chemotherapeutic agent, comprising a means for the isolation of protein or RNA from the tumor, a SPARC protein or RNA detection and quantification means, control RNAs, and rules for predicting the response of the tumor based on the level of SPARC protein or RNA in tumor.

FIELD OF THE INVENTION

This invention relates to methods for treating cancer; as well as otherdiseases involving abnormal proliferative, hyperplastic, remodeling,inflammatory activity in tissues and organs.

BACKGROUND OF THE INVENTION

Albumin nanoparticle formulations have been shown to reduce toxicity ofpoorly soluble therapeutic agents. For example, U.S. Pat. No. 6,537,579discloses an albumin-nanoparticle paclitaxel formulation which is freeof toxic emulsifiers.

The anticancer agent paclitaxel, marketed under the trademark Taxol® byBristol Myers Squibb, is currently approved for the treatment of severalcancers including ovarian, lung, and breast cancer. The major limitationto the use of paclitaxel is its poor solubility. Consequently, theTaxol® formulation contains Cremophor® EL as the solubilizing vehicle,but the presence of Cremophor® in this formulation has been linked tosevere hypersensitivity reactions in animals (Lorenz et al., AgentsActions 7, 63-67, 1987), and humans (Weiss et al., J. Clin. Oncol. 8,1263-1268, 1990). Accordingly, patients receiving Taxol® requirepremedication with corticosteroids (dexamethasone) and antihistamines toreduce the hypersensitivity and anaphylaxis that occurs due to thepresence of Cremophor®.

In contrast, Abraxane® also known as ABI-007 is a Cremophor®-free,albumin-nanoparticle formulation of paclitaxel, marketed by AbraxisOncology. The use of an albumin nanoparticle as a vehicle results in theformation of a colloid when reconstituted with saline. Based on clinicalstudies, it has been shown that the use of Abraxane® is characterized byreduced hypersensitivity reactions as compared with Taxol®. Accordingly,premedication is not required for patients receiving Abraxane®.

Another advantage of the albumin-nanoparticle formulation is that byexcluding toxic emulsifiers it is possible to administer higher doses ofpaclitaxel at more frequent intervals than is currently possible withTaxol®. The potential exists that enhanced efficacy could be seen insolid tumors as a consequence of (i) higher tolerable doses (300 mg/m²),(ii) longer half-life, (iii) prolonged local tumor availability and/or(iv) sustained in vivo release. Abraxane® reduces hypersensitivityreactions while maintaining or improving the chemotherapeutic effect ofthe drug.

It is known that colloidal nanoparticles or particles <200 nm in sizetend to concentrate at the tumor site due to leaky vasculatures. Thiseffect has been described for several lipsomal formulations(Papahadjopoulos, et al., Proc. Natl. Acad. Sci. U.S.A. 88, 11460,1991); (Gabizon, A., Cancer Res., 52, 891, 1992); (Dvorak, et al., Am.J. Pathol. 133, 95, 1988); (Dunn, et al., Pharm, Res., 11, 1016-1022,1994); and (Gref, et al, Science 263, 1600-1603, 1994). It is possiblethat localized nanoparticles of paclitaxel at the tumor site may resultin slow release of the drug at the tumor site resulting in greaterefficacy when compared to administration of the drug in its solubilized(Cremophor®-containing) form.

Such nanoparticle formulations comprise at least about 50% of the activeagent in nanoparticle form. Further, such nanoparticle formulationscomprise at least about 60%, at least about 70%, at least about 80%, orat least about 90% of the active agent in nanoparticle form. Moreover,such nanoparticle formulations comprise at least about 95% or at leastabout 98% of the active agent in nanoparticle form.

Secreted Protein, Acidic, Rich in Cysteines (SPARC), also known asosteonectin, is a 281 amino acid glycoprotein. SPARC has affinity for awide variety of ligands including cations (e.g., Ca²⁺, Cu²⁺, Fe²⁺),growth factors (e.g., platelet derived growth factor (PDGF), andvascular endothelial growth factor (VEGF)), extracellular matrix (ECM)proteins (e.g., collagen I-V and collagen IX, vitronectin, andthrombospondin-1), endothelial cells, platelets, albumin, andhydroxyapaptite. SPARC expression is developmentally regulated, and ispredominantly expressed in tissues undergoing remodeling during normaldevelopment or in response to injury (see, e.g., Lane et al., FASEB J.,8, 163-173 (1994)). High levels of SPARC protein are expressed indeveloping bones and teeth.

SPARC is a matricellular protein upregulated in several aggressivecancers, but is absent in normal tissues (Porter et al., J. Histochem.Cytochem., 43, 791 (1995)). Indeed, SPARC expression is induced among avariety of tumors (e.g., bladder, liver, ovary, kidney, gut, andbreast). In bladder cancer, for example, SPARC expression has beenassociated with advanced carcinoma. Invasive bladder tumors of stage T2or greater have been shown to express higher levels of SPARC thanbladder tumors of stage T1 (or less superficial tumors), and have poorerprognosis (see, e.g., Yamanaka et al., J. Urology, 166, 2495-2499(2001)). In meningiomas, SPARC expression has been associated withinvasive tumors only (see, e.g., Rempel et al., Clincal Cancer Res., 5,237-241 (1999)). SPARC expression also has been detected in 74.5% of insitu invasive breast carcinoma lesions (see, e.g., Bellahcene, et al.,Am. J. Pathol., 146, 95-100 (1995)), and 54.2% of infiltrating ductalcarcinoma of the breast (see, e.g., Kim et al., Korean Med. Sci., 13,652-657 (1998)). SPARC expression also has been associated with frequentmicrocalcification in breast cancer (see, e.g., Bellahcene et al.,supra), suggesting that SPARC expression may be responsible for theaffinity of breast metastases for the bone. SPARC is also known to bindalbumin (see, e.g., Schnitzer, J. Biol. Chem., 269, 6072 (1994)).

Antibody therapy is an effective method for controlling disease whereina specific protein marker can be identified. Examples include Avastin—ananti-VEGF antibody, Rituxan—an anti-CD20 antibody, and Remicade—ananti-TNF antibody. As such, an antibody against SPARC would represent animportant therapeutic agent for treating human and other mammaliantumors, as well as other proliferative, hyperplastic, remodeling, andinflammatory disorders that express the SPARC protein. In addition, anantibody against SPARC conjugated with an imaging or contrast agentwould be a means of detecting and diagnosing such disorders.

There remains a need for a method of treating human and other mammaliantumors, as well as other proliferative, hyperplastic, remodeling, andinflammatory disorders. Moreover, there remains a need for predicting ordetermining the response of a human or other mammalian tumor in order topredict or evaluate the effectiveness of the chemotherapeutic agent. Inaddition, suitable means are needed in order to detect and diagnose suchdisorders. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for predicting or determining theresponse of a mammalian tumor or other proliferative disease to achemotherapeutic agent or other anticancer agent, wherein the methodcomprises the steps of (a) isolating a biological sample from themammal, (b) detecting the expression of SPARC protein or RNA in thebiological sample, and (c) quantifying the amount of SPARC protein inthe biological sample. The invention provides for embodiments whereinthe biological sample is isolated from the tumor (or tissue involvedwith a proliferative disease) or from a bodily fluid, such as, e.g.,cerebrospinal fluid, blood, plasma, serum or urine.

In addition, the invention provides a method of treating a tumor orother proliferative disease in a mammal with a chemotherapeutic agent orother anticancer agent comprising (a) isolating a biological sample fromthe mammal, (b) detecting the expression of SPARC protein or RNA in thebiological sample, (c) quantifying the amount of SPARC protein or RNA inthe biological sample, (d) determining if the SPARC protein or RNA ispresent at a level indicating the use of the chemotherapeutic agent orother anticancer agent, and (e), if, based on the SPARC protein or RNAlevel, it is indicated, administering a therapeutically effective amountof the chemotherapeutic agent or other anticancer agent.

Further, the invention provides a kit for predicting the response of amammalian tumor or other proliferative disease to a chemotherapeuticagent or other anticancer agent, comprising a means for the isolation ofprotein from the tumor, a SPARC protein detection and quantificationmeans, control proteins, and rules for predicting the response of thetumor. The invention also provides for a kit for predicting the responseof a mammalian tumor or other proliferative disease to achemotherapeutic agent or other anticancer agent, comprising a means forthe isolation of RNA from the tumor, a SPARC RNA detection andquantification means, control RNAs, and rules for predicting theresponse of the tumor based on the level of SPARC RNA in tumor.

The invention provides for a method for predicting or determining theresponse of a mammalian tumor to a chemotherapeutic agent, as well as amethod of treating a mammalian tumor with a chemotherapeutic agent,wherein the chemotherapeutic agent is, e.g., docetaxel, paclitaxel, suchas Abraxane®, or combinations thereof. The invention further providesfor embodiments, wherein the prediction of the response of a mammaliantumor to a chemotherapeutic agent is positively or negatively correlatedwith SPARC levels.

The invention provides for a methods for predicting or determining theresponse of a mammalian tumor or treating a mammalian tumor with achemotherapeutic agent, where including, for example without limitation,wherein the mammal is a human and the tumor is a carcinoma of thebreast, ovary, head and neck, lung, colon, bladder or kidney.

The invention further provides a method for utilizing thealbumin-binding protein as a therapeutic, for utilizing SPARC andantibodies against SPARC or SPARC binding-proteins as therapeutics, fordelivering a chemotherapeutic agent to a disease site in a mammal, inwhich the method comprises administering to the mammal a therapeuticallyeffective amount of a pharmaceutical composition, wherein thepharmaceutical composition comprises the chemotherapeutic agent coupledto a compound capable of binding an albumin-binding protein and apharmaceutically acceptable carrier. In addition, the invention providesa composition comprising a chemotherapeutic agent coupled to a compoundcapable of binding a SPARC protein and a pharmaceutically acceptablecarrier. Moreover, the invention provides a delivery agent comprising aSPARC recognition group and a therapeutic agent, wherein the therapeuticagent is coupled to the SPARC recognition group. Furthermore, theinvention provides a method for delivering a chemotherapeutic agent to atumor in a mammal, wherein the method comprises administering to amammal a therapeutically effective amount of a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises achemotherapeutic agent coupled to a SPARC protein capable of bindingalbumin and a pharmaceutically acceptable carrier. The inventioncompositions may be small molecule, large molecule or proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the human SPARC amino acid sequence (SEQ ID NO: 1).

FIG. 2 depicts the human SPARC cDNA sequence (SEQ ID NO: 2).

FIG. 3 illustrates albumin and SPARC staining in MX-1 tumor xenografts.

FIG. 4 illustrates transcytosis of paclitaxel across endothelial cellmonolayers.

FIG. 5A-B illustrate the comparative efficacy (A, tumor volume) andtoxicity (B, % weight loss) of Abraxane and Taxotere for the treatmentof a MX-1 xenograft.

FIG. 6A-B illustrate the comparative efficacy (A, tumor volume) andtoxicity (B, % weight loss) of Abraxane and Taxotere for the treatmentof a LX-1 xenograft.

FIG. 7A-B illustrate the Her2 and SPARC status of the five tumor types(A) (SPARC, left bar and Her2, right bar in each pair) and the relativeefficacy of Abraxane versus Taxotere (B).

FIG. 8A-B illustrate the comparative efficacy (A, tumor volume) andtoxicity (B, % weight loss) of Abraxane and Taxotere for the treatmentof a HT29 xenograft.

FIG. 9A-B illustrate the comparative efficacy (A, tumor volume) andtoxicity (B, % weight loss) of Abraxane and Taxotere for the treatmentof a PC3 xenograft.

FIG. 10A-B illustrate the comparative efficacy (A, tumor volume) andtoxicity (B, % weight loss) of Abraxane and Taxotere for the treatmentof a MDA-MB-231 xenograft.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that an additional mechanism of localizationexists for compositions comprising albumin-binding proteins.Albumin-binding proteins, such as SPARC, cubilin, and TGFβ, can be usedto target a therapeutic agent to a disease site characterized by anoverexpression of an albumin-binding protein.

The invention provides for the use of a group, coupled to an agent,wherein the group is capable of binding an albumin-binding protein, suchas SPARC, cubilin, or TGFβ, and the agent is a therapeutic, imaging, ordelivery agent for diseases wherein the albumin-binding protein plays adominant role and is overexpressed relative to normal tissues.Preferably, the albumin-binding protein is selected from SPARC, cubilin,or TGFβ. Most preferred, the albumin-binding protein is SPARC and thegroup which binds the albumin-binding protein is a SPARC recognitiongroup. Suitable SPARC recognition groups include, but are not limited toligands, small molecules, antibodies.

The invention also provides a method for predicting or determining theresponse of a human or other mammalian tumor or other proliferativedisease to a chemotherapeutic agent or other anticancer agent. Themethod comprises (a) isolating a biological sample from the human orother mammalian, (b) detecting the expression of SPARC protein in thebiological sample, and (c) quantifying the amount of SPARC protein inthe biological sample. Once the amount of SPARC expressed by the tumoris determined, the effectiveness of the chemotherapeutic agent can bepredicted or ascertained by, for example, correlating of the expressionof SPARC to the dosage of therapeutic agent administered. The inventionalso provides for the use of antibody raised against SPARC as atherapeutic agent, or an imaging agent for diseases where SPARC plays adominant role and is overexpressed relative to normal tissues.

By “predicting the response of a human or other mammalian tumor or otherproliferative disease to a chemotherapeutic agent” it is meant making ajudgment, based on test results combined with clinical experience,regarding the likelihood of a response before administering thechemotherapeutic agent. By “determining the response of a human or othermammalian tumor to a chemotherapeutic agent” it is meant making ajudgment, based on test results combined with clinical experience,regarding the likelihood of a response after administering thechemotherapeutic agent but, before the response can be determinedclinically or by conventional laboratory or imagining studies known tothose of ordinary skill in the medical arts.

As used herein, “the response of a human or other mammalian tumor to achemotherapeutic agent” refers the degree or amount that the patientimproves clinically or that the tumor decreases in size oraggressiveness because of a chemotherapeutic agent. The patient can besaid to improve clinically based on objective criteria, such as, e.g.,performance status, physical examination, imaging studies or laboratorytest results. The patient also can be said to improve clinically basedsubjective criteria reported by the patient, such as, e.g., pain,distress, fatigue or mental outlook. Decreases in size tumor size can bebased on the primary tumor or overall tumor burden measured by anysuitable method known in the art, e.g., physical examination, imagingstudy or laboratory value. By “decrease in tumor size” it is meant achange of at least about 10%. Further, it is desirable that a change ofat least about 20% be present, preferably a change of at least about25%, more preferably a change of at least about 33%, more preferably achange of at least about 50%, more preferably a change of at least about90%, more preferably a change of at least about 95%, and most preferablya change of at least about 99%. By decrease in the “tumoraggressiveness” it is meant, e.g., a reduction the histologic grade, %viable cells in the tumor, % proliferating cells in the tumor, thetumor's invasiveness, the tumor's ability to metastasize or other metricof tumor aggressiveness know in the art. By “decrease in the tumor'saggressiveness” it is meant a change of at least about 10% in ameasurable parameter related to tumor aggressiveness which is commonlyused by those of ordinary skill in the medical arts. Further, it isdesirable that a change of at least about 20% be present, preferably achange of at least about 25%, more preferably a change of at least about33%, more preferably a change of at least about 50%, more preferably achange of at least about 90%, more preferably a change of at least about95%, and most preferably a change of at least about 99% in a measurableparameter related to tumor aggressiveness which is commonly used bythose of ordinary skill in the medical arts.

As used herein, the terms “resistant” or “resistance to achemotherapeutic or other anticancer agent” refers to an acquired ornatural resistance of a cancer sample or a mammal to a therapy, i.e.,being nonresponsive to or having reduced or limited response to thetherapeutic treatment, e.g., having a reduced response to a therapeutictreatment by 25% or more. Further, resistance can also be indicated by areduced response of, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more,to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more.The reduction in response is measured by comparing with the same cancersample or mammal before the resistance is acquired, or by comparing witha different cancer sample or a mammal who is known to have no resistanceto the therapeutic treatment. As used herein, the terms “sensitive” or“sensitive to a chemotherapeutic or other anticancer agent” refers tothe absence of resistance.

In addition, the invention provides for a method of treating a tumor orother proliferative disease in a mammal with a chemotherapeutic agent orother anticancer agent comprising: (a) isolating a biological samplefrom the mammal, (b) detecting the expression of SPARC protein or RNA inthe biological sample, (c) quantifying the amount of SPARC protein orRNA in the biological sample, (d) determining if the SPARC protein orRNA is present at a level indicating the use of the chemotherapeuticagent or other anticancer agent, and (e), if, based on the SPARC proteinor RNA level, it is indicated, administering a therapeutically effectiveamount of the chemotherapeutic agent or other anticancer agent.

The terms “treating,” “treatment,” “therapy,” and “therapeutictreatment” as used herein refer to curative therapy, prophylactictherapy, or preventative therapy. An example of “preventative therapy”is the prevention or lessening the chance of a targeted disease (e.g.,cancer or other proliferative disease) or related condition thereto.Those in need of treatment include those already with the disease orcondition as well as those prone to have the disease or condition to beprevented. The terms “treating,” “treatment,” “therapy,” and“therapeutic treatment” as used herein also describe the management andcare of a mammal for the purpose of combating a disease, or relatedcondition, and includes the administration of a composition to alleviatethe symptoms, side effects, or other complications of the disease,condition. Therapeutic treatment for cancer includes, but is not limitedto, surgery, chemotherapy, radiation therapy, gene therapy, andimmunotherapy.

By “determining if the SPARC protein or RNA is present at a levelindicating the use of the chemotherapeutic agent” it is meant that thequantified level of SPARC protein or RNA is present in the specimen fromthe mammal with a tumor is high enough, based on a comparison historicalcorrelation data of SPARC level and treatment response, to indicate thatthe tumor can be reasonably expected to respond to the chemotherapeuticagent. By “indicating” or “indicated” it meant that, in view of theSPARC level and based on reasonable medical judgment, thechemotherapeutic agent should be used. For example, without limitation,a biopsy of a tumor can be prepared for immunohistology with anti-SPARCantibodies by preparing a thin section of the biopsy on a microscopeslide. Then, the biopsy slide is stained using an anti-SPARCimmunohistological protocol (see, e.g., Sweetwyne et al., J. Histochem.Cytochem. 52(6):723-33 (2004); Tai et al., J. Clin. Invest.115(6):1492-502 (2005)) simultaneously with control slides containingsections of biopsies with known SPARC levels from other tumors sensitiveto and resistant to the chemotherapeutic agent considered for use. It isa common practice in the art to grade the intensity ofimmunohistological staining using light microscopy. The ordinarilyskilled artisan (e.g., a pathologist) can, based on comparison with thestaining of the control slides, assign a staining grade (e.g., 0, 1+,2+, 3+, 4+) to the tumor biopsy. Treatment with the chemotherapeuticagent can be “indicated” if the staining of the tumor biopsy is gradedat, e.g., 3+ or 4+. Such comparisons and assignments of staining gradesare well within the skill of the ordinarily skilled medical artisan(e.g., physician, pathologist, oncologist, veterinarian) treatingmammals with tumors.

By “therapeutically effective amount” it is meant an amount thatrelieves (to some extent, as judged by a skilled medical practitioner)one or more symptoms of the disease or condition in a mammal.Additionally, by “therapeutically effective amount” is meant an amountthat returns to normal, either partially or completely, physiological orbiochemical parameters associated with or causative of a disease orcondition. A clinician skilled in the art can determine thetherapeutically effective amount of a composition in order to treat orprevent a particular disease condition, or disorder when it isadministered, such as intravenously, subcutaneously, intraperitoneally,orally, or through inhalation. The precise amount of the compositionrequired to be therapeutically effective will depend upon numerousfactors, e.g., such as the specific activity of the active agent, thedelivery device employed, physical characteristics of the agent, purposefor the administration, in addition to many patient specificconsiderations. The determination of amount of a composition that mustbe administered to be therapeutically effective is routine in the artand within the skill of an ordinarily skilled clinician.

The methods practiced in accordance with the invention call for abiological sample which can be isolated from the tumor or tissuesinvolved with a proliferative disease by any suitable procedureincluding, without limitation, resection, biopsy, aspiration,venupuncture or combinations thereof. Alternatively, the methodspracticed in accordance with the invention call for a biological samplewhich can be from a bodily fluid, such as, e.g., cerebrospinal fluid,blood, plasma, serum, and urine. In addition, control or referencebiological samples including tumor and bodily fluid materials can beobtained from normal tissues of the same mammal, other individuals freeof tumor or proliferative disease or from other tumors with known SPARClevels and known to be sensitive to or resistant to a givenchemotherapeutic agent. Additionally, the methods of the invention canbe practiced wherein the mammal suffering from the tumor orproliferative disease is a human.

Further, the invention provides for a kit for predicting the response ofa mammalian tumor or other proliferative disease to a chemotherapeuticagent or other anticancer agent, comprising a means for the isolation ofprotein from the tumor, a SPARC protein detection and quantificationmeans, control proteins, and rules for predicting the response of thetumor. The invention also provides for a kit for predicting the responseof a mammalian tumor or other proliferative disease to achemotherapeutic agent or other anticancer agent, comprising a means forthe isolation of RNA from the tumor, a SPARC RNA detection andquantification means, control RNAs, and rules for predicting theresponse of the tumor based on the level of SPARC RNA in tumor. Forexample, the SPARC protein or RNA in a tumor biopsy can be “isolated” byplacing a thin section of the tumor biopsy on a microscope slide. AnySPARC protein or RNA present can then be detected and quantified byimmunohistological staining with an anti-SPARC antibody (see, e.g.,Sweetwyne et al., J. Histochem. Cytochem. 52(6):723-33 (2004); Tai etal., J. Clin. Invest. 115(6):1492-502 (2005)) or in situ hybridizationusing a nucleic acid probe complementary to SPARC RNA (see, e.g., Thomaset al., Clin. Can. Res. 6:1140-49 (2000)). At the same time positive andnegative control slides would be stained for SPARC protein or RNA. Theordinarily skilled artisan can readily use light microscopy to grade thestaining intensity of the SPARC in the tumor biopsy (e.g., 0, 1+, 2+,3+, 4+). The inventive kit also comprises rules for predicting theresponse of the tumor based on the level of SPARC protein or RNA intumor, such as, e.g., “treatment with the chemotherapeutic agent isindicated if the staining of the tumor biopsy is graded at, e.g., 3+ or4+” or “tumors with 3+ or 4+ staining have a high response rate.” Thespecific rules relating to a particular embodiment of the inventive kitscan readily be generated by performing retrospective or prospectivecorrelation studies which are routine in the art and which would notrequire undue experimentation.

The human SPARC gene encodes a 303 amino acid SPARC protein, whilemature SPARC is a 285 amino acid glycoprotein. After cleavage of thesignal sequence a 32-kD secreted form is produced which migrates at 43kD on SDS-PAGE because of glycosylation. The amino acid sequence of thecomplete SPARC protein is disclosed in SEQ ID NO: 1 (FIG. 1) and thenucleic acid sequence of an RNA encoding such a SPARC protein isdisclosed in SEQ ID NO: 2 (presented as a cDNA sequence, i.e., with theRNA uridines (“U”) as thymines (“T”)) (FIG. 2).

As used herein the terms “polypeptide” and “protein” are usedinterchangeably. The invention provides for the detection andquantification of a SPARC polypeptide or protein such as, e.g., apolypeptide or protein comprising an amino acid sequence of SEQ IDNO: 1. The invention also provides for the detection of SPARCpolypeptide, wherein the polypeptide comprises an amino acid sequence ofat least about 10 sequential amino acids from the sequence of SEQ ID NO:1, preferably at least about 15 sequential amino acids from the sequenceof SEQ ID NO: 1, more preferably at least about 20 sequential aminoacids from the sequence of SEQ ID NO: 1, and most preferably at leastabout 100 sequential amino acids from the sequence of SEQ ID NO: 1.Further, the invention provides for the detection of a SPARC polypeptidecomprising a polypeptide wherein the sequence is at least about 80%homologous to the corresponding sequence of SEQ ID NO: 1, preferably atleast about 90% homologous to the corresponding sequence of SEQ ID NO:1, even more preferably at least about 95% homologous to thecorresponding sequence of SEQ ID NO: 1, and even more preferably atleast about 99% homologous to the corresponding sequence of SEQ IDNO: 1. By “corresponding sequence of SEQ ID NO: 1” it is meant, thesequence which aligns with the sequence of SEQ ID NO: 1 wherein theregion of alignment is at least about 10 amino acids long, preferably isat least about 15 amino acids long, more preferably is at least about 20amino acids long, more preferably is at least about 30 amino acids long,more preferably is at least about 40 amino acids long, more preferablyis at least about 50 amino acids long, and even more preferably is atleast about 100 amino acids long. Various methods of sequence alignmentare known in the biotechnology arts (see, e.g., Rosenberg, BMCBioinformatics 6:278 (2005); Altschul et al., FEBS J. 272(20): 5101-5109(2005)).

The invention provides for the detection and quantification of a SPARCRNA such, e.g., an RNA comprising the nucleic acid sequence of SEQ IDNO: 2. The invention also provides for the detection of SPARC RNA,wherein the RNA comprises the nucleic sequence of at least about 15sequential nucleotides from the sequence of SEQ ID NO: 2, preferably atleast about 20 sequential nucleotides from the sequence of SEQ ID NO: 2,and more preferably at least about 30 sequential nucleotides from thesequence of SEQ ID NO: 2. Further, the invention provides for thedetection of a SPARC RNA comprising a nucleic acid wherein the sequenceis at least about 80% homologous to the corresponding sequence of SEQ IDNO: 2, preferably at least about 90% homologous to the correspondingsequence of SEQ ID NO: 2, even more preferably at least about 95%homologous to the corresponding sequence of SEQ ID NO: 2, and even morepreferably at least about 99% homologous to the corresponding sequenceof SEQ ID NO: 2. By “corresponding sequence of SEQ ID NO: 2” it ismeant, the sequence which aligns with the sequence of SEQ ID NO: 2wherein the region of alignment is at least about 15 nucleotides long,preferably is at least about 20 nucleotides long, more preferably is atleast about 30 nucleotides long, more preferably is at least about 60nucleotides long, more preferably is at least about 120 nucleotideslong, more preferably is at least about 150 nucleotides long, even morepreferably is at least about 200 nucleotides long. Various methods ofsequence alignment are known in the biotechnology arts (see, e.g.,Rosenberg, BMC Bioinformatics 6:278 (2005); Altschul et al., FEBS J.272(20): 5101-5109 (2005)). By SPARC RNA it is meant any SPARC RNA,including but, not limited to, a SPARC mRNA, hnRNA, primary transcriptor splice variant.

By “quantification” as used herein it is meant determining the amount orconcentration present. The invention provides for a method ofquantifying the level of SPARC protein or RNA wherein SPARC protein orRNA is overexpressed or underexpressed in the tumor relative to normaltissues, including but, not limited to, the level found in thecorresponding normal tissue of origin of the tumor. Alternatively, Theinvention provides for a method of quantifying the level of SPARCprotein or RNA wherein SPARC protein or RNA is overexpressed orunderexpressed in the tumor relative to other tumors, including but notlimited to, tumors of the same tissue or histology. Further, Theinvention provides for a method of quantifying the level of SPARCprotein or RNA wherein SPARC protein or RNA is overexpressed orunderexpressed in the tumor relative to other tumors, including but, notlimited to, tumors which are sensitive to or resistant to achemotherapeutic agent or combination of chemotherapeutic agents. Byoverexpressed or underexpressed it is meant that the levels of SPARCprotein or RNA differs between the two specimens or samples by at leastabout 5%. Further, it is desirable that the difference between the twospecimens or samples is at least about 10%, more preferably at leastabout 20%, more preferably at least about 50%, more preferably at leastabout 100%, more preferably at least about 3 fold, more preferably atleast about 5 fold, and most preferably at least about 10 fold.

The invention provides for a method of quantifying the level of SPARCprotein or RNA wherein SPARC protein or RNA is overexpressed orunderexpressed in the test biological fluid relative to correspondingfluid from a tumor-free patient. Alternatively, The invention providesfor a method of quantifying the level of SPARC protein or RNA whereinSPARC protein or RNA is overexpressed or underexpressed in the testbiological fluid relative to corresponding fluid from a another patientwith a tumor, including but not limited to, tumors which are sensitiveto or resistant to a chemotherapeutic agent or combination ofchemotherapeutic agents. By overexpressed or underexpressed it is meantthat the levels of SPARC protein or RNA differs in two specimens by atleast about 5%. Further, it is desirable that a difference of at leastabout 10% is present, preferably at least about 20%, more preferably byat least about 50%, more preferably by at least about 100%, morepreferably at least about 3 fold, more preferably at least about 5 fold,and most preferably at least about 10 fold.

The invention provides methods of predicting or determining a tumor'sresponse to a chemotherapeutic agent or other anticancer agents, methodsof treating a tumor, and kits for predicting the response of a mammaliantumor to a chemotherapeutic agent or other anticancer agent, wherein thetumor is selected from the group consisting of oral cavity tumors,pharyngeal tumors, digestive system tumors, the respiratory systemtumors, bone tumors, cartilaginous tumors, bone metastases, sarcomas,skin tumors, melanoma, breast tumors, the genital system tumors, urinarytract tumors, orbital tumors, brain and central nervous system tumors,gliomas, endocrine system tumors, thyroid tumors, esophageal tumors,gastric tumors, small intestinal tumors, colonic tumors, rectal tumors,anal tumors, liver tumors, gall bladder tumors, pancreatic tumors,laryngeal tumors, tumors of the lung, bronchial tumors, non-small celllung carcinoma, small cell lung carcinoma, uterine cervical tumors,uterine corpus tumors, ovarian tumors, vulvar tumors, vaginal tumors,prostate tumors, prostatic carcinoma, testicular tumors, tumors of thepenis, urinary bladder tumors, tumors of the kidney, tumors of the renalpelvis, tumors of the ureter, head and neck tumors, parathyroid cancer,Hodgkin's disease, Non-Hodgkin's lymphoma, multiple myeloma, leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloidleukemia, chronic myeloid leukemia. In addition, the invention providesfor method of predicting or determining a tumor's response to achemotherapeutic agent, methods of treating a tumor, and kits forpredicting the response of a mammalian tumor to a chemotherapeuticagent, wherein the tumor is a sarcoma, adenocarcinoma, squamous cellcarcinoma, large cell carcinoma, small cell carcinoma, basal cellcarcinoma, clear cell carcinoma, oncytoma or combinations thereof.Further, the invention provides for method of predicting or determininga tumor's response to a chemotherapeutic agent, methods of treating atumor, and kits for predicting the response of a mammalian tumor to achemotherapeutic agent, wherein the tumor is a benign tumor or amalignant tumor. Yet further, the invention provides for method ofpredicting or determining a proliferative disease's response to achemotherapeutic agent or treating a proliferative disease, includingbut, not limited to, where the proliferative diseases is, e.g., benignprostatic hyperplasia, endometriosis, endometrial hyperplasia,atherosclerosis, psoriasis or a proliferative renal glomerulopathy. Theinvention provides for embodiments wherein the tumor or proliferativedisease is in mammal including but, not limited to, where the mammal isa human.

As used herein, the term “agent” or “drug” or “therapeutic agent” refersto a chemical compound, a mixture of chemical compounds, a biologicalmacromolecule, or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues that are suspected of having therapeutic properties. The agentor drug may be purified, substantially purified or partially purified.An “agent”, according to the present invention, also includes aradiation therapy agent. As used herein, the term “chemotherapueticagent” refers to an agent with activity against cancer, neoplastic,and/or proliferative diseases.

Suitable chemotherapeutic agents or other anticancer agents for use inaccordance with the invention include but, are not limited to, tyrosinekinase inhibitors (genistein), biologically active agents (TNF, of tTF),radionuclides (¹³¹I, ⁹⁰Y, ¹¹¹In, ²¹¹At, ³²P and other known therapeuticradionuclides), adriamycin, ansamycin antibiotics, asparaginase,bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine,chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine,dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin,etoposide, epothilones, floxuridine, fludarabine, fluorouracil,gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin(sirolimus) and derivatives, mitomycin, mitotane, mitoxantrone,nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin,procarbazine, rituximab, streptozocin, teniposide, thioguanine,thiotepa, taxanes, vinblastine, vincristine, vinorelbine, taxol,combretastatins, discodermolides, and transplatinum. Accordingly,suitable chemotherapeutic agents for use in accordance with inventioninclude, without limitation, antimetabolites (e.g., asparaginase),antimitotics (e.g., vinca alkaloids), DNA damaging agents (e.g.,cisplatin), proapoptotics (agents which induce programmed-cell-death orapoptosis) (e.g., epipodophylotoxins), differentiation inducing agents(e.g., retinoids), antibiotics (e.g., bleomycin), and hormones (e.g.,tamoxifen, diethylstibestrol). Further, suitable chemotherapeutic agentsfor use in accordance with the invention include antiangiogenesis agents(angiogenesis inhibitors) such as, e.g., INF-alpha, fumagillin,angiostatin, endostatin, thalidomide, and the like. “Other anticanceragents” also include, without limitation, biologically activepolypeptides, antibodies, lectins, and toxins. Suitable antibodies foruse in accordance with the invention include, without limitation,conjugated (coupled) or unconjugated (uncoupled) antibodies, monoclonalor polyclonal antibodies, humanized or unhumanized antibodies, as wellas Fab′, Fab, or Fab2 fragments, single chain antibodies and the like.

Preferred chemotherapeutic agents include docetaxel, paclitaxel, andcombinations thereof. “Combinations thereof” refers to both theadministration of dosage forms including more than one drug, forexample, docetaxel and paclitaxel, as well as the sequential but,temporally distinct, administration of docetaxel and paclitaxel (e.g.,the use of docetaxel in one cycle and paclitaxel in the next).Particularly preferred chemotherapeutic agents comprise particles ofprotein-bound drug, including but not limited to, wherein the proteinmaking up the protein-bound drug particles comprises albumin includingwherein more than 50% of the chemotherapeutic agent is in nanoparticleform. Most preferably the chemotherapeutic agent comprises particles ofalbumin-bound paclitaxel, such as, e.g., Abraxane®. Such albumin-boundpaclitaxel formulations can be used in accordance with the inventionwhere the paclitaxel dose administered is from about 30 mg/m² to about1000 mg/m² with a dosing cycle of about 3 weeks (i.e., administration ofthe paclitaxel dose once every about three weeks). Further, it isdesirable that the paclitaxel dose administered is from about 50 mg/m²to about 800 mg/m², preferably from about 80 mg/m² to about 700 mg/m²,and most preferably from about 250 mg/m² to about 300 mg/m² with adosing cycle of about 3 weeks.

Any suitable biological sample can be isolated from the mammal in thecontext of the inventive method and used for polypeptide and/or RNAdetection and quantification. Preferably, the biological sample isisolated from the tumor, such as by a tumor biopsy. The biologicalsample is isolated from the mammal using methods known in the art.Alternatively, the biological sample can be isolated from a bodily fluidof the mammal, including, for example, cerebrospinal fluid, blood,plasma, serum, or urine. In particular, many protein purificationtechniques are known in the art (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor, pp. 421-696 (1988)).

Any suitable method for the detection and quantification of a SPARCprotein can be used in accordance with the invention including, but notlimited to, the use of anti-SPARC antibodies (e.g., Western blot, ELISA)(see, e.g., Sweetwyne et al., J. Histochem. Cytochem. 52(6):723-33(2004); Tai et al., J. Clin. Invest. 115(6):1492-502 (2005)), the use ofSPARC-specific binding proteins (e.g., radiolabel SPARC ligands,ELISA-like assays), two-dimensional electrophoresis, mass spectroscopyor combinations thereof (see, e.g., Nedelkov D et al., Proc. Natl. Acad.Sci. U.S.A. 102(31):10852-7 (2005); Chen et al., Proc. Natl. Acad. Sci.U.S.A. 101(49):17039-44 (2004)). Further, immunohistochemistry can beused for the isolation, detection and quantification of SPARC protein ina sample (see, e.g., Sweetwyne et al., J. Histochem. Cytochem.52(6):723-33 (2004); Tai et al., J. Clin. Invest. 115(6):1492-502(2005)).

The invention provides for a method wherein the SPARC RNA is detectedand quantified. Numerous methods are known in the art to isolate RNA,such as the ones described by Chomczynski (U.S. Pat. No. 5,945,515) orby DiMartino et al. (Leukemia 20(3):426-32 (2006)). Alternatively, RNAcan be isolated in a form suitable for detection and quantification inaccordance with the invention by the preparation of a microscope slidecontaining a tissue section (see, e.g., Thomas et al., Clin. Can. Res.6:1140-49 (2000)). SPARC RNA can be detected and quantified by anysuitable method known in the art including but, not limited to, in situhybridization (see, e.g., Thomas et al., Clin. Can. Res. 6:1140-49(2000)), Northern blot (see e.g., Wrana et al., Eur. J. Biochem.197:519-28 (1991)), real-time RT-PCR (see, e.g., DiMartino et al.,Leukemia 20(3):426-32 (2006)), Real-time nucleic acid sequence-basedamplification (see, e.g., Landry et al., J. Clin. Microbiol.43(7):3136-9 (2005)), microarray analysis (see, e.g., Tai et al., J.Clin. Invest. 115(6):1492-502 (2005); DiMartino et al., Leukemia20(3):426-32 (2006)) and combinations thereof.

The invention also provides a method for predicting or determining theresponse of a human or other mammalian tumor or other proliferativedisease to a chemotherapeutic agent or other anticancer agents whereinthe response of a mammalian tumor to a chemotherapeutic agent ispositively or negatively correlated with SPARC levels. By “correlatedwith SPARC levels” it is meant, e.g., that a mutual or reciprocalrelation between the tumor's response to a given chemotherapeutic agentand the level of SPARC protein or RNA detected. That is, the quality,degree, magnitude, or level of the tumor response varies with the levelof the level of SPARC protein or RNA detected. A “positive correlation”is present when the quality, degree, magnitude, or level of the tumorresponse increases as the level of SPARC protein or RNA detectedincreases. A “negative correlation” is present when the quality, degree,magnitude, or level of the tumor response decreases as the level ofSPARC protein or RNA detected increases. The relation between the levelthe tumor response and the level of SPARC protein or RNA detected cantake on the form of or approximate a step-function, linear-function orlogarithmic function.

In addition, the invention also provides a method for predicting ordetermining the response of a human or other mammalian tumor or otherproliferative disease to a chemotherapeutic agent or other anticanceragents by comparing the level of SPARC protein or RNA detected to thatdetected in a known reference sample. Such a reference sample can befrom, for example, a normal tissue or bodily fluid. Alternatively, thereference sample can be a tumor with a known SPARC level, response,sensitivity or resistance to a given chemotherapeutic agent or otheranticancer agents or combinations thereof.

Further, the predicted response can be characterized as effective or asnot effective such that a given chemotherapeutic agent would be used oran alternative chemotherapeutic agent would be used. As such, thepredicted response can be characterized as a ratio of the responseresulting from the use of one chemotherapeutic agent versus the use ofanother chemotherapeutic agent, e.g., the ratio of the response producedby Abraxane® to that produced by Taxotere®.

The invention further provides methods of predicting the response of amammalian tumor or other proliferative disease to a chemotherapeuticagent or other anticancer agent comprising (a) isolating a biologicalsample from the mammal, (b) detecting the expression of SPARC protein orRNA in the biological sample, (c) quantifying the amount of SPARCprotein or RNA in the biological sample, and (d) determining the Her2status of the tumor or proliferative disease. The invention alsoprovides methods of treating a tumor or other proliferative disease in amammal with a chemotherapeutic agent or other anticancer agentcomprising: (a) isolating a biological sample from the mammal, (b)detecting the expression of SPARC protein or RNA in the biologicalsample, (c) quantifying the amount of SPARC protein or RNA in thebiological sample, (d) determining the Her2 status of the tumor orproliferative disease, (e) determining if, based on the SPARC protein orRNA level present and the Her2 status of the tumor or proliferativedisease, whether the use of the chemotherapeutic agent or otheranticancer agent is indicated, and (f), if indicated, administering atherapeutically effective amount of the chemotherapeutic agent or otheranticancer agent.

By “determining the Her2 status of the tumor or proliferative disease”it is meant determining whether or not Her2 protein or RNA is expressedby the tumor or other proliferative disease by detecting the expressionof Her2 protein or RNA in the tumor, tissue involved by anotherproliferative disease or other biological sample and quantifying theamount of Her2 protein or RNA present. Suitable methods for determiningwhether Her2 protein or RNA is expressed by a tumor or proliferativedisease include, without limitation, immunohistochemistry, Western blot,ELISA, two-dimensional electrophoresis, mass spectroscopy, in situhybridization, RNA amplification, Northern blot, microarray analysis orcombinations thereof. Her2 status can be factored into predicting aresponse to chemotherapuetic or other anticancer agent or determiningwhether a chemotherapuetic or other anticancer agent is indicated eitherpositively or negatively, as an additive factor or as a predominantfactor. For example, without limitation, the absence of Her2 expressioncan indicate that there is no positive correlation between SPARC leveland response, while the presence of Her2 expression can indicate thatthere is a positive correlation between SPARC level and response. Thedistinction between Her2 expression and its absence is within the skillof those of ordinary skill in the art and can be made based on, e.g.,the results of positive and negative control samples run simultaneouslywith the samples from the mammal with the tumor or other proliferativedisease.

Accordingly, the invention provides for a kit for predicting theresponse of a mammalian tumor or other proliferative disease to achemotherapeutic agent or other anticancer agents, comprising a meansfor the isolation of protein or RNA from the tumor, a SPARC protein orRNA detection and quantification means, control proteins or RNAs, andrules for predicting the response of the tumor. Such a kit can, forexample without limitation, be used to predict the response of a breast,ovarian or head and neck carcinoma to a chemotherapeutic agentcomprising nanoparticles of albumin-bound paclitaxel. Suitable means forisolating protein or RNA and a SPARC protein or RNA detection andquantification have been described herein. Suitable control proteins orRNAs should include positive controls such as, e.g., tumor material orbiological fluid from a tumor bearing mammal or isolated protein or RNAfrom tumor material or from a biological fluid harvested from a tumorbearing mammal. Suitable control proteins or RNAs include negativecontrols such as, e.g., natural tissue or biological fluid from a mammalfree of tumor or isolated protein or RNA from normal tissue orbiological fluid harvested from a mammal free of tumor. Controls in thekit can also include materials use to establish standard curves forquantification of SPARC protein or RNA or material from sensitive andresistant tumors. The kits of the invention can also comprising a meansfor determining the Her2 status of the tumor.

The inventive kits would further comprise rules for predicting theresponse of the tumor. Such rules would base the prediction of responseto a given chemotherapeutic agent on the level of SPARC protein or RNAdetected as described herein in relation to the methods of predicting ordetermining a response to chemotherapeutic agent. For example, aparticular level of SPARC protein or RNA, based on past experience, canindicate that a chemotherapeutic agent should be used. It is within theskill of the ordinarily skilled artisan to generate, without undueexperimentation, adequate data (by prospective studies, retrospectivestudies or a combination thereof) to determine the level of SPARCprotein or RNA predictive of response to a given chemotherapeutic agent.

Hereinafter, for simplicity, all proteins, including SPARC, that bindalbumin are referred to as SPARC. The SPARC protein is responsible forthe accumulation of albumin in certain human tumors. As albumin is themajor carrier of chemotherapeutic drugs, the expression level of SPARCis indicative of the amount of chemotherapeutic drug that penetrates andis retained by the tumor. Therefore, the expression level of SPARC ispredictive of the responsiveness of the tumor to chemotherapy.

Any suitable biological sample can be isolated from the mammal ofinterest in the context of the inventive method. Preferably, thebiological sample is isolated from the tumor, such as by a tumor biopsy.Alternatively, the biological sample can be isolated from a bodily fluidof the mammal, including, for example, cerebrospinal fluid, blood,plasma, serum, or urine. Techniques and methods for the isolation ofbiological samples are known to those in the art.

Any suitable pharmaceutically active agent can be used in the inventivemethod (e.g., a chemotherapeutic agent coupled to a SPARC recognitiongroup), so long as the transport or binding of the active agent requiresalbumin. Suitable active agents include, but are not limited to,tyrosine kinase inhibitors (genistein), biologically active agents (TNFor tTF), radionuclides (e.g., ¹³¹I, ⁹⁰Y, ¹¹¹In, ²¹¹At, ³²P, and otherknown therapeutic radionuclides), adriamycin, ansamycin antibiotics,asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine,capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin,dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel,doxorubicin, etoposide, epothilones, floxuridine, fludarabine,fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine, mechlorethamine, mercaptopurine, meplhalan,methotrexate, rapamycin (sirolimus) and derivatives, mitomycin,mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate,pentostatin, plicamycin, procarbazine, rituximab, streptozocin,teniposide, thioguanine, thiotepa, taxanes, vinblastine, vincristine,vinorelbine, taxanes, combretastatins, discodermolides, transplatinum,vascular targeting agents, anti-vascular endothelial growth factorcompounds (“anti-VEGFs”), anti-epidermal growth factor receptorcompounds (“anti-EGFRs”), 5-fluorouracil, and derivatives thereof.

In addition, the pharmaceutically active agent can be toxins such asricin A, radionuclides, the Fc fragment of the antibody itself, singlechain antibodies, Fab fragments, diabodies, and the like. Thepharmaceutically active agents selected themselves can recognize andbind to SPARC or are suitably attached to a SPARC recognition group thatrecognizes SPARC, including, for example, a protein or non-protein, anantibody, Fc fragment of the antibody itself, single chain antibodies,Fab fragments, diabodies, peptides, or other non-protein smallmolecules.

One or more doses of one or more chemotherapeutic agents can beadministered according to the inventive methods. The type and number ofchemotherapeutic agents used in the inventive method will depend on thestandard chemotherapeutic regimen for a particular tumor type. In otherwords, while a particular cancer may be treated routinely with a singlechemotherapeutic agent, another may be treated routinely with acombination of chemotherapeutic agents. Methods for coupling orconjugation of suitable therapeutics, chemotherapeutics, radionuclides,etc. to antibodies or fragments thereof are well described in the art.

Diseases for which the present invention is useful include abnormalconditions of proliferation, tissue remodeling, hyperplasia, exaggeratedwound healing in any bodily tissue including soft tissue, connectivetissue, bone, solid organs, blood vessel and the like. Examples ofdiseases treatable or diagnosed by invention compositions includecancer, diabetic or other retinopathy, inflammation, arthritis,restenosis in blood vessels or artificial blood vessel grafts orintravascular devices and the like.

The types of tumor to be detected, whose response to chemotherapy is tobe predicted or determined, which can be treated in accordance with theinvention are generally those found in humans and other mammals. Thetumors can be the result of inoculation as well, such as in laboratoryanimals. Many types and forms of tumors are encountered in human andother animal conditions, and there is no intention to limit theapplication of the methods of the present to any particular tumor typeor variety. Tumors, as is known, include an abnormal mass of tissue thatresults from uncontrolled and progressive cell division, and is alsotypically known as a “neoplasm.” The inventive methods are useful fortumor cells and associated stromal cells, solid tumors and tumorsassociated with soft tissue, such as, soft tissue sarcoma, for example,in a human. The tumor or cancer can be located in the oral cavity andpharynx, the digestive system, the respiratory system, bones and joints(e.g., bony metastases), soft tissue, the skin (e.g., melanoma), breast,the genital system, the urinary system, the eye and orbit, the brain andcentral nervous system (e.g., glioma), or the endocrine system (e.g.,thyroid) and is not necessarily limited to the primary tumor or cancer.Tissues associated with the oral cavity include, but are not limited to,the tongue and tissues of the mouth. Cancer can arise in tissues of thedigestive system including, for example, the esophagus, stomach, smallintestine, colon, rectum, anus, liver, gall bladder, and pancreas.Cancers of the respiratory system can affect the larynx, lung, andbronchus and include, for example, non-small cell lung carcinoma. Tumorscan arise in the uterine cervix, uterine corpus, ovary vulva, vagina,prostate, testis, and penis, which make up the male and female genitalsystems, and the urinary bladder, kidney, renal pelvis, and ureter,which comprise the urinary system. The tumor or cancer can be located inthe head and/or neck (e.g., laryngeal cancer and parathyroid cancer).The tumor or cancer also can be located in the hematopoietic system orlymphoid system, and include, for example, lymphoma (e.g., Hodgkin'sdisease and Non-Hodgkin's lymphoma), multiple myeloma, or leukemia(e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyeloid leukemia, chronic myeloid leukemia, and the like). Preferably,the tumor is located in the bladder, liver, ovary, kidney, gut, brain,or breast.

The SPARC protein has affinity for a wide variety of ligands. Thus, theinventive method for delivering a therapeutic agent to a site of diseaseis predicated on the discovery that compounds or ligands, includingalbumin, which have affinity for SPARC can be used to delivertherapeutic drugs to the site of disease, with little or no delivery tonormal tissues.

The invention also provides for a means of transporting the therapeuticcomposition across the endothelial barrier from the blood vessel intothe tumor interstitium. The main hurdle in antibody therapy andchemotherapy is the translocation across the endothelial barrier intotumor interstitium. Albumin utilizes the albumin receptor transportmechanism to cross the endothelial barrier. This transport mechanismcould be the same as those reported by the literature (gp60 andalbondin) or by other undiscovered mechanisms. It has been previouslyreported that the therapeutic agent piggy backed onto albumin exhibitedenhanced tumoral uptake (Desai, N. et al. Increased endothelialtranscytosis of nanoparticle albumin-bound paclitaxel (ABI-007) byendothelial gp60 receptors: a pathway inhibited by Taxol®, 27^(th)Annual San Antonio Breast Cancer Symposium (SABCS) (2004), abstract#1071). Further, enhanced translocation across the endothelial barriercan be achieved using the physiological albumin transport mechanism(Schnitzer, J. E.; Oh, P. J. Biol. Chem. 269, 6072-6082 (1994).

For small molecules, modifications can be made so that the drug affinityfor albumin is increased. For formulations of small molecules, a solventwhich prevents the binding of the drug to albumin may be removed.Alternatively, the small molecule may be linked to albumin, antibodyagainst albumin, fragments thereof or ligands for an albumin-receptorsuch as described below.

For biologic molecules such as proteins, antibodies and fragmentsthereof, it is possible to engineer the biologics with an albuminbinding peptide such that the biologics will exhibit an affinity foralbumin. The peptide can either be an albumin binding sequence, anantibody or antibody fragment against albumin, antibody or antibodyfragment against albumin carriers (such as gp60/albondin/scavengerreceptor/or TGF-beta receptor), or antibody to any of the proteins foundin the caveolae, the transporter of albumin. The invention alsocontemplates an antibody or suitable fragment thereof prepared as achimera with one valence for SPARC and another valence for an effectorof transendothelial transport such as gp60/albondin/scavengerreceptor/or TGF-beta receptor, or against any of the proteins found inthe caveolae of the endothelial cell.

The invention also provides a method for the destruction of SPARCexpression tissues such as tumor and restenotic tissues via thecomplement fixation and/or recruitment of cell mediated immune responseby SPARC antibody. In this case, like that of Rituxan, an anti-CD20antibody, the effector moiety is the Fc fragment which can mediateeither complement activation with direct destruction of SPARC expressioncells or recruitment of immune cells to the SPARC expression tissue withresulting tissue destruction via a cell mediated immune response.

The invention also provides a method for inhibition of SPARC activityusing a neutralizing antibody against SPARC. The neutralizing antibodyhas the ability to block the interaction of SPARC with its effectors invivo. For example, the neutralizing antibody may block interaction ofSPARC with a cell surface component or the binding of SPARC to itsnatural ligands such as albumin, growth factors, and Ca²⁺.

The invention also provides a method for determining the response of ahuman or other mammalian tumor to anti-SPARC therapy. The methodcomprises (a) isolating a biological sample from the human, (b)detecting the expression of SPARC protein in the biological sample, and(c) quantifying the amount of SPARC protein in the biological sample. Asanti-SPARC therapy relies on the binding of SPARC antibody to SPARC indisease tissue, the presence of SPARC in disease tissue is necessary foractivity.

The invention further provides a method of using one or more diagnosticagents conjugated to the SPARC recognition groups, such as theantibodies or fragments thereof, as described above. The diagnosticagents include radioisotopes or radionuclides, MRI contrast agents,X-ray contrast agents, ultrasound contrast agents and PET contrastagents. Methods utilized for conjugation are known in the art.

The expression of SPARC protein in a sample can be detected andquantified by any suitable method known in the art. Suitable methods ofprotein detection and quantification include Western blot, enzyme-linkedimmunosorbent assay (ELISA), silver staining, the BCA assay (Smith etal., Anal. Biochem., 150, 76-85 (1985)), the Lowry protein assay(described in, e.g., Lowry et al., J. Biol. Chem., 193, 265-275 (1951)),which is a colorimetric assay based on protein-copper complexes, and theBradford protein assay (described in, e.g., Bradford et al., Anal.Biochem., 72, 248 (1976)), which depends upon the change in absorbancein Coomassie Blue G-250 upon protein binding. Tumor biopsy can beanalyzed by any of the preceeding methods or it can be analyzed byimmunohistochemistry using anti-SPARC antibody (either monoclonal orpolyclonal) in conjunction with appropriate visualization system (i.e.,HRP substrate and HRP-conjugated secondary antibody).

Any suitable antibodies against SPARC can be used in the inventivemethod, so long as the antibody exhibits specific binding to SPARC. Theantibody can either be monoclonal or polyclonal; and can be producedeither through immunization of an animal or produced through recombinantDNA technology such as phage display and in vitro mutagenesis orsynthesis of the variable regions of the antibody heavy and light chaingenes. Polyclonal antibodies include, but are not limited to humanantibodies and humanized antibodies derived from animals such as avian(e.g., chicken), rodent (e.g., rat, mouse, hamster, guinea pig), cow,goat, sheep, rabbit and the like. Monoclonal antibodies includeantibodies derived from a single clone of antibody producing cellsincluding, but not limited to, human cells, and antibodies derived fromthe cells of other animal types, for example, chicken, rabbit, rat,mouse, hamster, guinea pig, cow, goat, sheep, and the like. Syntheticantibodies include antibodies produced using recombinant DNA technologyvia genetic engineering of the variable regions of the heavy and lightchain genes. Synthetic antibodies also include chemically synthesizedantibody fragments with SPARC binding activity or antibodies derivedfrom phage display or similar technology.

For human use, in order to avoid immunogenicity and immune response, itis preferable to use humanized anti-SPARC antibody or suitable fragmentssuch as Fab′, Fab, or Fab2. Humanized antibody or fragments thereofmaybe produced, for example, using one of the following establishedmethods: 1) humanized antibody may be constructed using human IgGbackbone replacing the variable CDR region with that of antibody againstSPARC, where the heavy and light chain are independently expressed underseparate promoters or coexpressed under one promoter with IRES sequence;2) humanized monoclonal antibody may be raised against SPARC using amouse engineered to have a human immune system; 3) humanized antibodyagainst SPARC may be raised using phagemid (M13, lambda coliphage, orany phage system capable of surface presentation). To construct the fulllength antibody, the variable region may be transferred onto the CDR ofboth Heavy chain and Light chain. The coexpression of the Heavy chainand Light Chain in mammalian cells such as CHO, 293, or human myeloidcells results in full length antibody. Similarly, Fab′, Fab, or Fab2fragments and single chain antibodies can be prepared using wellestablished methods.

Antibody against SPARC is also not limited to whole antibody or fragmentof the antibody retaining the binding site for SPARC (e.g., Fab andFab2). The antibody is also not limited to any one class of antibody,e.g., IgM, IgA, IgG, IgE, IgD, and IgY. The antibody is also not limitedto divalent antibody, monovalent, or chimera with one valence for SPARCand another for an effector such tTF or ricin A. The humanized antibodyis not limited to IgG. The same technologies can be used to generate allother classes of antibodies such as IgE, IgA, IgD, IgM, each havingdifferent antibody-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) activities appropriate to particulardisease target. Functional fragments of the antibody can be generated bylimited proteolysis. These fragments can be monovalent such as Fab′ ordivalent, such as Fab2. Fragments can also be synthesized as singlechain scfv or diabodies in E. coli.

The invention further provides a composition comprising apharmaceutically active agent directly able to exert its pharmacologicaleffect or a pharmaceutically active agent coupled to a compound capableof binding SPARC, or other or other albumin binding moiety, and apharmaceutically acceptable carrier. The delivery agent, which may be apharmaceutical composition, comprise the pharmaceutically active agentcoupled to a SPARC recognition group is administered to a mammal, suchas a human, in an amount such that a therapeutically effective amount ofthe pharmaceutically active agent is delivered to the mammal.

The invention also provides a method for delivering a chemotherapeuticagent to a tumor in a mammal. The method comprises administering to ahuman or other mammal a therapeutically effective amount of apharmaceutical composition, wherein the pharmaceutical compositioncomprises the chemotherapeutic agent coupled to a compound or ligandcapable of binding a SPARC protein and a pharmaceutically acceptablecarrier. Descriptions of the chemotherapeutic agent, tumor, mammal, andcomponents thereof, set forth herein in connection with otherembodiments of the invention also are applicable to those same aspectsof the aforesaid method of delivering a chemotherapeutic agent to atumor.

Preferably, the pharmaceutical composition does not comprise more than50% of the therapeutic agent in nanoparticle form. More preferably, thepharmaceutical composition does not comprise more than 10% of thetherapeutic agent in nanoparticle form. Even more preferably, thepharmaceutical composition does not comprise more than 5%, or more than4% or more than 3% of the therapeutic agent in nanoparticle form. In amore preferred embodiment, the pharmaceutical composition does notcomprise more than 2% or, more than 1% of the therapeutic agent innanoparticle form. Most preferably, the pharmaceutical composition doesnot comprise any of the therapeutic agent in nanoparticle form.

The invention also provides a method for delivering a chemotherapeuticagent to a tumor in a human or other mammal. The method comprisesadministering to a human or other mammal a therapeutically effectiveamount of a delivery agent, such as a pharmaceutical composition,wherein the delivery agent (e.g., pharmaceutical composition) comprisesthe chemotherapeutic agent coupled to the SPARC recognition group. Forexample, the chemotherapeutic agent can be coupled to a SPARCrecognition group such as an antibody recognizing SPARC protein, or theSPARC antibody alone. Pharmaceutical compositions preferably include thechemotherapeutic agent coupled to the SPARC recognition group and apharmaceutically acceptable carrier. Descriptions of thechemotherapeutic agent, tumor, mammal, and components thereof, set forthherein in connection with other embodiments of the invention also areapplicable to those same aspects of the aforesaid method of delivering achemotherapeutic agent to a tumor.

In other embodiments, the invention provides a method for delivering apharmaceutically active agent by way of a SPARC recognition group to asite of disease that is characterized by overexpression of SPARC oranother albumin-binding protein or marker in a human, or other animalthat expresses such protein or marker. Such diseases include abnormalconditions of proliferation, tissue remodeling, hyperplasia, andexaggerated wound healing in bodily tissue (e.g., soft tissue,connective tissue, bone, solid organs, blood vessel and the like).Examples of diseases that are treatable or may be diagnosed byadministering a pharmaceutical composition comprising a therapeuticagent coupled to a compound or ligand capable of binding a SPARCprotein, or another albumin-binding protein, include cancer, diabetic orother retinopathy, inflammation, arthritis, restenosis in blood vessels,artificial blood vessel grafts, or intravascular devices, and the like.Descriptions of the pharmaceutically active agent, tumor, mammal, andcomponents thereof, set forth herein in connection with otherembodiments of the invention also are applicable to those same aspectsof the aforesaid method of delivering a pharmaceutically active agent.

In yet other embodiments, the invention provides a method for deliveringa pharmaceutically active agent (for example, SPARC antibody alone orchemotherapeutic agent conjugated to SPARC recognition group such asSPARC antibody, radiolabelled SPARC antibody and the like) to a site ofdisease that is characterized by overexpression of SPARC in a human, orother animal that expresses such protein or marker. Such diseasesinclude abnormal conditions of proliferation, tissue remodeling,hyperplasia, and exaggerated wound healing in bodily tissue (e.g., softtissue, connective tissue, bone, solid organs, blood vessel and thelike). Examples of diseases that are treatable or diagnosed byadministering a pharmaceutical composition comprising anti-SPARCtherapy, include cancer, diabetic or other retinopathy, inflammation,arthritis, restenosis in blood vessels, artificial blood vessel grafts,or intravascular devices, and the like. Descriptions of thepharmaceutically active agent, tumor, mammal, and components thereof,set forth here in connection with other embodiments of the inventionalso are applicable to those same aspects of the aforesaid method ofdelivering a pharmaceutically active agent.

In other embodiments, the inventive method comprises administering to amammal a therapeutically effective amount of a pharmaceuticalcomposition comprising a chemotherapeutic agent coupled to a compound orligand capable of binding SPARC protein. The chemotherapeutic agent canbe coupled to the compound or ligand capable of binding SPARC proteinusing any suitable method. Preferably, the chemotherapeutic agent ischemically coupled to the compound via covalent bonds including, forexample, disulfide bonds.

The invention also provides a method comprising administering to amammal a therapeutically effective amount of a pharmaceuticalcomposition comprising a chemotherapeutic agent or radioactive elementcoupled to a SPARC recognition group. The chemotherapeutic orradioactive agent can be coupled to the antibody recognizing SPARC usingany suitable method. Preferably, the chemotherapeutic agent can bechemically coupled to the compound via covalent bonds including, forexample, disulfide bonds.

Preferably, the compound or ligand useful in the inventive method iscapable of binding the SPARC protein. In a preferred embodiment of theinvention, the compound is a ligand that binds a SPARC protein. Examplesof suitable ligands include calcium cation (Ca²⁺), copper cation (Cu²⁺),iron cation (Fe²⁺), platelet derived growth factor (PDGF), vascularendothelial growth factor (VEGF), collagen (e.g., collagen I, collagenII, collagen III, collagen IV, collagen V, and collagen IX),vitronectin, thrombospondin-1, endothelial cells, platelets, albumin,and hydroxyapatitecations. In another preferred embodiment of theinvention, the compound is a small molecule. The term “small molecule”refers to any molecule having a molecular weight less than about 600.Examples of suitable small molecules include a protein, a nucleic acid,a carbohydrate, a lipid, a coenzyme (e.g., a vitamin), an antigen, ahormone, and a neurotransmitter. Preferably, the small molecule is achemical (e.g., an organic or inorganic chemical) a peptide, or peptidemimetic, a protein, or a carbohydrate. In yet another preferredembodiment of the invention, the compound is an antibody that isdirected against a SPARC protein. Any suitable antibody, or fragmentthereof, that binds to a SPARC protein can be used in the inventivemethod.

SPARC expression in tumor tissues has been demonstrated in almost allcancer types. There has been evidence that elaboration of SPARC on tumortissues can be either derived from tumoral expression of SPARC or bystromal cells. The SPARC phenotype of the tumor can be converted fromSPARC negative to SPARC positive by administration of exogenous SPARC.Accordingly, the SPARC positive tumor would then become sensitive tochemotherapeutic agents. Alternatively, SPARC could be radiolabeled orconjugated with various toxins to confer on it the ability to kill thetumor directly or indirectly.

SPARC can be synthesized and purified using known technologies. Cellsexpressing exogenous SPARC can be generated by placing the SPARCstructural gene/cDNA under the control of strong promoter/translationstart and the vector transfected into mammalian cells to drive theexpression of SPARC in these cells. Alternatively, SPARC can beexpressed using bacculovirus or other viruses such as adenovirus. SPARCexpressed by these cells can be purified by traditional purificationsmethods such as ionic exchange, size exclusion, or C18 chromatography.The purified SPARC can be formulated in saline with preservatives andadministered intravenously, by aerosol, by subcutaneous injection, orother methods.

The invention also provides a method for delivering a chemotherapeuticagent to a tumor in a mammal. The method comprises administering to amammal a therapeutically effective amount of a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises thechemotherapeutic agent coupled to a SPARC protein capable of bindingalbumin and a pharmaceutically acceptable carrier. Descriptions of thechemotherapeutic agent, tumor, mammal, and components thereof, set forthherein in connection with other embodiments of the invention also areapplicable to those same aspects of the aforesaid method of delivering achemotherapeutic agent to a tumor.

For use in vivo, the chemotherapeutic agent coupled to a compound orligand capable of binding the SPARC protein desirably is formulated intoa pharmaceutical composition comprising a physiologically acceptablecarrier. Any suitable physiologically acceptable carrier can be usedwithin the context of the invention, and such carriers are well known inthe art.

For use in vivo, the anti-SPARC therapy agent desirably is formulatedinto a pharmaceutical composition comprising a physiologicallyacceptable carrier. Any suitable physiologically acceptable carrier canbe used within the context of the invention, and such carriers are wellknown in the art.

The carrier typically will be liquid, but also can be solid, or acombination of liquid and solid components. The carrier desirably is aphysiologically acceptable (e.g., a pharmaceutically orpharmacologically acceptable) carrier (e.g., excipient or diluent).Physiologically acceptable carriers are well known and are readilyavailable. The choice of carrier will be determined, at least in part,by the location of the target tissue and/or cells, and the particularmethod used to administer the composition.

Typically, such compositions can be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for using toprepare solutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and the preparations can also beemulsified. The pharmaceutical formulations suitable for injectable useinclude sterile aqueous solutions or dispersions; formulationscontaining known protein stabilizers and lyoprotectants, formulationsincluding sesame oil, peanut oil or aqueous propylene glycol, andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the formulation must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxycellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The chemotherapeutic agent (e.g., anti-SPARC therapy) coupled to acompound or ligand which binds a SPARC protein can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such as organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups also can be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The composition can further comprise any other suitable components,especially for enhancing the stability of the composition and/or itsend-use. Accordingly, there is a wide variety of suitable formulationsof the composition of the invention. The following formulations andmethods are merely exemplary and are in no way limiting.

Formulations suitable for administration via inhalation include aerosolformulations. The aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also can be formulated as non-pressurizedpreparations, for delivery from a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. In a preferred embodiment of theinvention, the chemotherapeutic agent coupled to a compound which bindsa SPARC protein (e.g., anti-SPARC therapy) is formulated for injection(e.g., parenteral administration). In this regard, the formulationdesirably is suitable for intratumoral administration, but also can beformulated for intravenous injection, intraperitoneal injection,subcutaneous injection, and the like.

Formulations suitable for anal administration can be prepared assuppositories by mixing the active ingredient with a variety of basessuch as emulsifying bases or water-soluble bases. Formulations suitablefor vaginal administration can be presented as pessaries, tampons,creams, gels, pastes, foams, or spray formulas containing, in additionto the active ingredient, such carriers as are known in the art to beappropriate.

In addition, the composition can comprise additional therapeutic orbiologically-active agents. For example, therapeutic factors useful inthe treatment of a particular indication can be present. Factors thatcontrol inflammation, such as ibuprofen or steroids, can be part of thecomposition to reduce swelling and inflammation associated with in vivoadministration of the pharmaceutical composition and physiologicaldistress.

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope.

Example 1

This example illustrates the co-localization of SPARC with albumin in anMX-1 tumor xenograft.

Paclitaxel albumin nanoparticles (Abraxane, ABX or ABI-007) have beenshown to have an improved response rate over Taxol (TAX) in a Phase 3metastatic breast cancer trial (33% vs. 19%, p<0.0001) (see, e.g.,O'Shaughnessy, SABCS). Albumin-mediated transendothelial transport ofpaclitaxel (P) and increased intratumoral accumulation of paclitaxel forABX versus TAX was demonstrated recently (see, e.g., Desai, SABCS 2003).Albumin binds to SPARC (see, e.g., Schnitzer, J. Biol. Chem., 269,6072-82 (1994)).

The MX-1 tumor cell line is derived from a human breast cancer. Serialcryosections of human MX-1 tumor xenograft, human primary breast tumortissues (n=141), and normal human breast tissue (n=115) wereimmunostained and scored (0-4) for albumin, SPARC (using anti-SPARCantibody), and caveolin-1 staining. Cultured MX-1 cells also wereimmunostained for SPARC. Paclitaxel albumin nanoparticles (Abraxane, ABXor ABI-007) and Taxol (TAX) were prepared with radioactive paclitaxel(P) (20 mg/kg IV), and were used to determine the biodistribution ofpaclitaxel in normal tissues of athymic mice.

Albumin staining in the MX-1 tumor was focal and co-localized with SPARC(FIG. 3). Caveolin-1 staining confirmed that blood vessel density inalbumin-containing areas was no different from albumin-free areas. SPARCexpression by MX-1 cultured cells was confirmed by positive stainingwith anti-SPARC antibody. Paclitaxel accumulation in normal tissues(SPARC negative) was significantly lower for ABX as compared to TAX(p<0.004) for 7/10 tissues. 46% of the human primary breast tumorsexhibited strong SPARC staining (score >2), as compared to 1% for normaltissues (p<0.0001). In a subset of 50 tumor tissues, SPARC expressiondid not correlate with staging, ER status, or PgR status; however, therewas trend for high SPARC expression among p53-negative tumors.

The co-localization of albumin and SPARC suggests that SPARC, by itsalbumin binding activity, may behave as an intratumoral target foralbumin binding in breast tumors. As transport of paclitaxel in ABX isdependent on albumin (see, e.g., Desai SABCS, 2003), this may explainthe improved tumor accumulation of ABX as compared to TAX. ABXaccumulation in normal tissues was lower than for TAX, consistent withlack of SPARC expression in normal tissues. Screening of patients forSPARC allows for the identification of patients more responsive to ABX.The presence of SPARC in these tumors allows for targeting and therapyusing anti-SPARC antibody.

Example 2

This example illustrates the expression of SPARC in MX-1 tumor cells.

MX-1 cells were cultured on a coverslip and stained with an antibodydirected against human SPARC using methods known in the art. Antibodystaining was observed, which demonstrates that MX-1 is expressing SPARC.These results suggest that SPARC expression detected in MX-1 tumor cellsis a result of SPARC secretion by MX-1 tumor cells. Staining was moreintense for MX-1 tumor cells than that of normal primary cells such asHUVEC (human umbiblical vein endothelial cells), HLMVEC (Human lungmicrovessel endothelial cells), and HMEC (Human mammary epithelialcells). Though the majority of the SPARC staining was internal SPARC,significant level of surface SPARC was detected as demonstrated byconfocal miscroscopy and staining of unpermeabilized cells.

Example 3

This example illustrates the overexpression of SPARC protein in humanbreast carcinoma cells.

SPARC expression in human breast carcinoma cells was determined using atumor array from Cybrdi, Inc. (Gaithersburg, Md.). The results of thisanalysis are set forth in Table 1. Intensity of staining was scored from“Negative” to 4+, with the higher number corresponding to greaterintensity of overexpression. 49% of breast carcinoma stained positive(2+ and above) for SPARC, as compared to 1% of normal tissue (p<0.0001).

SPARC Staining (%) Negative −/+ 1+ 2+ 3+ 4+ Carcinoma 31 14 1 11 9 25Cells (34%) (15%) (1%) (12%) (10%) (27%) Normal Cells 93  7 4  1 0  0(89%)  (7%) (4%)  (1%)  (0%)  (0%)

Example 4

This example illustrates endothelial receptor (gp60)-mediated caveolartranscytosis of paclitaxel albumin nanoparticles (ABI-007).

Paclitaxel (P) albumin nanoparticles (Abraxane, ABX or ABI-007)demonstrated improved response rate over Taxol in a phase III metastaticbreast cancer trial (33% vs 19%, p<0.0001) (SABCS, O'Shaughnessy et al,2003). Cremophor in Taxol (TAX) entraps P in micelles in plasma,reducing the paclitaxel available for cellular partitioning (see, e.g.,Sparreboom et al., Cancer. Res., 59, 1454 (1999)). Studies in athymicmice have shown 30-40% higher intratumor paclitaxel concentrations withABX as compared to equal doses of TAX (SABCS, Desai et al, 2003).Albumin is transported across endothelial cells (EC) by specificreceptor (gp60)-mediated caveolar transport (see, e.g., John et al., Am.J. Physiol., 284, L187 (2001)). It was hypothesized that albumin-boundpaclitaxel in ABX may be transported across tumor microvessel EC bygp60, and this mechanism may be particularly active for ABX as comparedto TAX.

A series of experiments were performed to evaluate binding and transportof paclitaxel by human umbilical vein endothelial cells (HUVEC) andhuman lung microvessel endothelial cells (HLMVEC) for ABX and TAX.Fluorescent paclitaxel (FP) was used as a probe and fluorescent ABX andTAX were formulated with FP to probe the binding and transport ofpaclitaxel across EC monolayers grown on a transwell apparatus.

Binding of paclitaxel to cells (HUVEC) was 10× higher for ABX than TAX.The transport of paclitaxel from ABX across EC monolayers was enhancedby 2-3 fold and 2-4 fold for HUVEC and HMVEC, respectively, as comparedto TAX. Transport was dependent on albumin. Transport of paclitaxel fromABX was inhibited by the presence of anti-SPARC antibody, which is knownto bind gp60, the receptor required for caveolar albumin transcytosis.Known inhibitors of calveolar transcytosis, NEM and β-methylcyclodextrin (BMC), also inhibited the transport of paclitaxel from ABXacross the endothelial monolayers (FIG. 4). Inhibition of caveolartransport decreased transport of P from ABX to the level of TAXtransport.

These results demonstrate that paclitaxel from ABX is activelytransported across EC by gp60-mediated caveolar transcytosis, whereas Pfrom TAX appears to be transported at a 2-4 fold lower rate primarily bya paracellular (non-caveolar) mechanism. This pathway may in part beresponsible for increased intratumoral concentrations of paclitaxel seenfor ABX relative to TAX. Cremophor in TAX inhibits transcytosis ofpaclitaxel across endothelial cells.

Example 5

This example demonstrates correlation of SPARC overexpression with highresponse rates using nanoparticle albumin-bound paclitaxel (ABI-007) insquamous head and neck cancers.

In phase I and II clinical studies of patients with squamous cellcarcinoma (SCC) of head and neck (H&N) and anal canal, response rates of78% and 64% were observed, respectively, for intra-arterially deliveredNanoparticle Albumin-Bound Paclitaxel (Abraxane®, ABX or ABI-007) (see,e.g., Damascelli et al., Cancer, 92(10), 2592-2602 (2001), andDamascelli et al., AJR, 181, 253-260 (2003)). In comparing in vitrocytoxicity of ABX and Taxol (TAX), we observed that a squamous cervix(A431) line demonstrated improved IC₅₀s for ABX (0.004 μg/ml) vs TAX(0.012 μg/ml). Albumin-mediated transendothelial caveolar transport ofpaclitaxel (P) and increased intratumoral accumulation of P for ABX vsTAX was demonstrated recently (see, e.g., Desai, SABCS 2003).

Human H&N tumor tissues (n=119) and normal human H&N tissue (n=15) wereimmunostained and scored (0-4) for SPARC staining using a tumor andnormal tissue array. Immunostaining was performed using polyclonalrabbit anti-SPARC antibody. In a new phase I dose escalation study (ABXgiven IV over 30 minutes q3w), a subset of head and neck cancer patients(n=3) were analyzed for response to ABX.

SPARC was overexpressed (score >2) in 60% (72/119) of the H&N tumorsversus 0% (0/15) in normal tissues (p<0.0001). In the phase I study, 2/3H&N patients achieved partial response (PR) after 2 cycles of treatmentat dose levels of 135 mg/m² (1 pt) and 225 mg/m² (1 pt). A third patientat 260 mg/m² progressed.

SPARC was found to be overexpressed in 60% of squamous H&N tumors. Thismay explain the high single-agent activity of ABX seen previously insquamous H&N cancers due to binding of albumin-bound paclitaxel to SPARCexpressed in these tumors. 2/3 patients with squamous H&N tumorsachieved PR in a new phase I study.

SPARC Staining: - --------------  0 −/+ 1 2 3 4 H&N Tumor Array:Carcinoma 17 14 16  23  20  29  - -------------- (14%) (12%) (13%) (19%)(17%) (24%) Normal 13  0 2 0 0 0 - -------------- (87%)  (0%) (13%) (0%)  (0%)  (0%)Human H&N tumor tissues (n=119) and normal human H&N tissue (n=15) wereimmunostained and scored (0-4) for SPARC staining using a tumor andnormal tissue array. Immunostaining was performed using polyclonalrabbit anti-SPARC antibody. SPARC was overexpressed (score >2) in 60%(72/119) of the H&N tumors versus 0% (0/15) in normal tissues(p<0.0001). This may explain the high single-agent activity of ABX seenpreviously in squamous H&N cancers due to binding of albumin-boundpaclitaxel to SPARC expressed in these tumors.

In a new phase I dose escalation study (ABX given IV over 30 minutesq3w), a subset of head and neck cancer patients (n=3) were analyzed forresponse to ABX. In the phase I study, 2/3 H&N patients achieved partialresponse (PR) after 2 cycles of treatment at dose levels of 135 mg/m² (1pt) and 225 mg/m² (1 pt). A third patient at 260 mg/m² progressed. Tumortissues from these patients were stained for SPARC and 1 respondingpatients showed strong overexpression for SPARC.

In another phase II clinical study of 54 patients with squamous cellcarcinoma of head and neck treated with intra-arterial ABX, an overallresponse rate of 78% was noted. Cancer biopsies from 16 patients in thisstudy receiving intra-arterial ABX were evaluated for SPARC expressionand correlation with clinical response. Staining with anti-SPARCpolyclonal antibody (R&D Systems, Minneapolis, Minn., USA) was scored ona 0-4 scale (032 no staining, 4=strong positive). Positive SPARCexpression was identified as >2+ staining and negative SPARC expressionwas identified as <2+ staining. The ABX—responders exhibited higherincidence of SPARC expression (10/11, 91%) versus nonresponders (2/5,40%) (p=0.06.) ABX—response was significantly higher for SPARC-positivepatients (10/12=83%) versus SPARC-negative patients (1/4=25%) (p=0.06).In addition, the SPARC-negative patients exhibited significantly lowerresponse rate than the overall response rate in the study (includingpatients treated with ABX or other chemotherapeutic agents) (1/4, 25%vs. 42/54, 78%; p<0.05)).

Example 6

This example illustrates the internalization of labeled albumin intoMIX-1 tumor cells and colocalization within the MIX-1 cell withintracellular SPARC expression.

MX-1 cells were cultured on a coverslip and permeabilized with suitableagents. Cells were exposed to fluorescent albumin and following washingwere exposed to SPARC antibody. This was followed by exposure to asecondary antibodies having a different fluorescent tag than thealbumin. It was surprisingly observed that the labeled albumincolocalised with the presence of SPARC within the cell indicating thatalbumin was rapidly internalized and targeted intracellular SPARC.

Example 7

This example demonstrates an increase in endothelial transcytosis viagp60 (albumin receptor) of pharmaceutical compositions comprisingpaclitaxel and albumin as compared to Taxol.

Human lung microvessel endothelial cells (HLMVEC) were grown toconfluence on a transwell. The inventive pharmaceutical compositioncomprising paclitaxel and albumin, or Taxol containing fluorescentpaclitaxel (Flutax) at a concentration of 20 μg/mL, was added to theupper transwell chamber.

The transport of paclitaxel by transcytosis from the upper chamber tothe lower chamber was monitored continuously using a fluorometer. Acontrol containing only Flutax without albumin was also used. Thecontrol with Flutax showed no transport, validating the integrity of theconfluent HLMVEC monolayer. Transport of paclitaxel from thealbumin-paclitaxel composition was much faster than paclitaxel fromTaxol in the presence of 5% HSA (physiological concentration). Transportrate constants (K_(t)) for the albumin-paclitaxel composition and Taxolwere 1.396 h⁻¹ and 0.03 h⁻¹, respectively. The total amount ofpaclitaxel transported across the monolayer was three times higher forthe albumin-paclitaxel composition than Taxol. Thus, the use of albuminor other suitable mimetic including antibodies or fragments against thegp60 receptor or other endothelial cell receptor can assist in thetransport of a desired therapeutic agent across the endothelial barrierinto the tumor interstitium.

Example 8

This example demonstrates the specific binding of anti-SPARC antibody toSPARC.

Whole cell extract was prepared from HUVEC cells by sonication. Theprotein was separated on a s5-15% SDS-PAGE, transferred onto PVDFmembrane and visualized with a polyclonal antibody against SPARC and amonoclonal antibody against SPARC. Both antibodies reacted to a singleband at 38 kDa, the correct molecular weight for SPARC. When MX-1 wasanalyzed by the same method, SPARC was detected in both the clarifiedcell lysate or the membrane rich membrane fraction.

Example 9

This example demonstrates the absence of SPARC expression in normaltissues.

Normal human and mouse tissue were immunostained and scored (0-4) forSPARC staining using a tumor and normal tissue array. Immunostaining wasperformed using polyclonal rabbit anti-SPARC antibody. SPARC was notexpressed in any of the normal tissues, with the exception of theesophagus. Likewise, SPARC was not expressed in any of the normal mousetissue, except the kidney of the female mouse. However, it is possiblethat this expression was due to follistatin which is homologous toSPARC.

SPARC Expression in Human Normal Tissues

Stomach 0/8 Colon 0/9 Rectum 0/15 Liver 0/14 Spleen 0/10 Lung 0/14Kidney 1/14 Brain 1/14 Testis 0/8 Prostate 0/3 Heart 0/9 Tonsil 0/10Lymph Nodes 0/10 Appendix 0/10 Esophagus 5/5 Pancreas 0/5 Eyeball 0/5Ovary 0/5

Mouse Normal Tissues

Liver 0/19 Kidney (M) 0/8 Kidney (F) 6/8 Lung 0/16 Muscle 0/20 Brain0/20 Heart 0/18 Stomach 0/20 Spleen 0/20

Example 10

This example demonstrates the superiority of Abraxane (nanoparticlealbumin-bound paclitaxel, “ABX”) to Taxotere (docetaxel formulated withTween 80 and ethanol, “TAT”) and that the relative response to ABXpositively correlates with SPARC expression in the Her2-positivexenografts tested.

A murine tumor xenograft model system was used to compare the efficacyof ABX versus TAT in Her2-negative xenografts of the MX-1 human breastcarcinoma and LX-1 human lung carcinoma cell lines. In the MX-1 study 8animals were used in each of three groups: TAT (once per week for threeweeks, iv) at 15 mg/kg; ABX (once per week for three weeks, iv) at 15mg/kg, and saline (once per week for three weeks, iv). Each animal'sweight and tumor size obtained weekly for three weeks. In the LX-1 study8 animals were used in each of three groups: TAT (once every four daysfor three cycles, iv) at 15 mg/kg; ABX (once every four days for threecycles, iv) at 50 mg/kg or 120 mg/kg, and saline (once every four daysfor three cycles, iv). Each animal's weight and tumor size obtainedweekly for three weeks.

Xenograft tumor Her2 and SPARC status were determined byimmunohistochemistry using a monoclonal anti-human Her2 antibody and apolyclonal anti-human SPARC antibody. The slides were scored using a 0-4scale, 0 being negative and 4 being strong positive.

In the MX-1 study, ABX at equidose was more effective than TAT(p<0.0001, ANOVA statistic) (see FIG. 5A). For the LX-1 cells, ABX wasmore effective than TAT (15 mg/kg) at both dose levels (p=0.0001 for ABX50 mg/kg and p<0.0001 for ABX 120 mg/kg) (see FIG. 6A). However, ABXresponse did not correlate with SPARC level by immunohistochemistry (seeFIG. 7).

In a second study, a murine tumor xenograft model system was used tocompare the Efficacy of ABX versus TAT in three Her2-positive xenografts(HT29: human colonic carcinoma cell line, PC3: human prostate carcinomacell line, MDA-MB-231: human breast carcinoma cell line). Thirty twomice were used for each xenograft with eight animals treated with TAT(once every four days for three cycles, iv) at 15 mg/kg, eight animalstreated with ABX (once every four days for three cycles, iv) at 50mg/kg, eight animals treated with ABX (once every four days for threecycles, iv) at 120 mg/kg, and eight control animals treated with saline(once every four days for three cycles, iv). Each animal's weight andtumor size obtained for each of the three weeks.

The Her2 and SPARC status were determined by immunohistochemistry usinga monoclonal anti-human Her2 antibody and a polyclonal anti-human SPARCantibody. The slides were scored using a 0-4 scale, 0 being negative and4 being strong positive.

In the HT29 xenograft, ABX was more effective than TAT (15 mg/kg) atboth dose levels (p=0.0057 for ABX 50 mg/kg and p<0.0001 for ABX 120mg/kg) (see FIG. 8A). With the PC3 xenograft, ABX was less effectivethan TAT (15 mg/kg) at 50 mg/kg (p<0.001) but, equally effective as TAT(15 mg/kg) at 120 mg/kg (p=ns) (FIG. 9A) and less toxic (less weightloss, FIG. 9B). Finally, with the MDA-MB-231 xenograft, ABX was lesseffective than TAT (15 mg/kg) at both dose levels (p<0.0001 andp<0.0001) (see FIG. 10A).

The Her2 and SPARC status of the five tumor types is shown in FIG. 7Aand the relative efficacy of ABX versus TAX is shown in FIG. 7B.Relative efficacy (TAT tumor volume/ABX tumor volume) was calculatedusing 15 mg/kg TAT and 120 mg/kg ABX, (except for MX-1 where therelative tumor efficacy was calculated using 15 mg/kg TAX and 15 mg/kgABX.) In the Her2-positive xenografts (HT29, PC3, and MDA-MB-231), ABXefficacy relative to TAT increased with increasing SPARC expression (seeFIG. 7).

Thus, this example shows that, in the model system used, in four of fivexenograft tumors tested, ABX was more effective or equally effectivecompared to TAT and that, in the Her2-positive xenografts tested, ABXefficacy relative to TAX is positively correlated with SPARC expression.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-45. (canceled)
 46. A method of treating a tumor of the breast,prostate or colon in a mammal with a chemotherapeutic agent or otheranticancer agent comprising: (a) isolating a biological sample from themammal, (b) detecting the expression of SPARC protein in the biologicalsample, (c) quantifying the amount of SPARC protein in the biologicalsample to determine whether the biological sample is positive for SPARCexpression, (d) detecting the expression of Her2 protein in thebiological sample, (e) quantifying the amount of Her2 protein in thebiological sample to determine if the biological sample is positive forHer2 expression, and (f) if the biological sample is positive for SPARCexpression and positive for Her2 expression, administering atherapeutically effective amount of the chemotherapeutic agent or otheranticancer agent.
 47. The method of claim 46, wherein thechemotherapeutic agent is select from the group consisting of docetaxel,paclitaxel, taxanes, platinum compounds, antifolates, antimetabolites,intimitotics, DNA damaging agents, proapoptotics, differentiationinducing agents, antiangiogenic agents, antibiotics, hormones, peptides,and combinations thereof.
 48. The method of claim 46, wherein thechemotherapeutic agent comprises particles of protein-bound drug. 49.The method of claim 48, wherein the protein component of theprotein-bound drug particles comprises albumin.
 50. The method of claim46, wherein the chemotherapeutic agent comprises particles ofalbumin-bound paclitaxel.
 51. The method of claim 50, wherein more than50% of the chemotherapeutic agent is in nanoparticle form.
 52. Themethod of claim 51, wherein the paclitaxel dose is from about 30 mg/m2to about 1000 mg/m² with a dosing cycle of about 3 weeks.
 53. The methodof claim 46, wherein the mammal is a human.